Abstract: In this study, we established a mild alkali-ethanol extraction method for the extraction and refinement of crocodile oil from cultured crocodile fat. The refined oil was obtained at a yield of 75% of the fat materials, without unfavorable and fishy smell; GC-MS analysis indicated that the refined oil contains 25 kinds of fatty acid and the unsaturated fatty acids accounted for 65.4% of oil, and is free of lead, mercury and arsenic. This work provides a simple and efficient method for large-scale preparation of high quality crocodile oil.